Optical membrane formation system and method

ABSTRACT

The invention relates to a spectroscopy system and improved membrane formation techniques. The system has at least one light source operable to generate a source beam and a prism having a rear surface. A support block is disposed on the rear surface of the prism. The support block is formed with at least one sample well having a center and defines a substantially vertical rear cell surface having a center. The source beam is aimed at the sample well. A syringe filled with a membrane solution and having a needle with a distal end disposed in front of the sample well. The distal end is aimed at a point above the center of the rear cell surface. The syringe is operable to eject a steady stream of membrane solution from the needle onto the circular rear cell surface thereby forming a membrane defining at least a portion of a layer under test. The membrane has a substantially uniform thickness that covers substantially the entire rear cell surface. A detector operable to detect light that is at least one of reflected and scattered by the layer under test.

[0001] This application claims the priority of U.S. ProvisionalApplication 60/400,183 filed Aug. 1, 2002.

[0002] The invention relates to the field of membrane formation and inparticular relates to the formation of optical membranes on opticalsurfaces such as prisms and the like. The invention is useful inconnection with various spectroscopy techniques operable to characterizesurface phenomenon including but not limited to: fluorescence microscopysystems, low angle neutron scattering systems, X-Ray scattering systems,surface plasmon resonance systems, plasmon-waveguide resonance systemsand the like.

[0003] In the case of surface plasmon resonance systems, alight-reflecting surface is coated with a thin metallic coating. Lightat a specific incident angle excites the electrons in the metal. Thisresults in localized fluctuations of the electron density known assurface plasmons. The light energy transferred to the metal coatingduring excitation results in an attenuation of the reflected lightintensity. The incident angle and degree of the attenuation depends onthe wavelength of the exciting light and the thickness and opticalproperties of the interface in contact with the metal coating.

[0004] The important optical properties of such an interface include theabsorbance at the excitation wavelength (extinction coefficient), therefractive index, and the thickness of the interface. The effectivedistance of Surface Plasmon penetration is only several hundrednanometers (nm) so only the environment at the surface is detected. Thisproperty makes Surface Plasmon Resonance (SPR) and Plasmon WaveguideResonance (PWR) ideal for measuring surface and interfacial chemistry,as well as the properties of thin film coating properties (includingmolecular films). The formation of such films or membranes haspreviously been carried out manually. See e.g., U.S. Pat. No.5,521,702—Salamon, et al., discussed below.

[0005] It is also understood that gold and silver are two metals thatproduce strong SPR signals. Under similar conditions the SPR electricfield in the sample produced by silver is more than 2 times strongerthan gold resulting in much sharper resonances and greater sensitivity.However, the chemical reactivity of silver renders it inappropriate formany applications. Therefore many applications utilize gold as themetallic coating.

[0006] PWR is essentially a species of SPR however, PWR techniquesutilize one or more dielectric coatings (e.g., silica dioxide) over themetallic coating. The appropriate dielectric coatings serve as both ashield and an “optical amplifier”. PWR allows the use of silver as themetallic coating or layer, with its improved optical properties butwithout suffering from its undesirable chemical properties.

[0007] SPR systems utilize specific light polarizations (e.g.,p-polarization) in reference to the sample plane to produce resonancesignals. In PWR systems, the appropriate dielectric coating also servesas an optical amplifier resulting in additional sharpening of theresonance spectrum, and more importantly, allowing light polarizationsboth parallel (s-polarization) and perpendicular (p-polarization) to thesample plane to produce resonance signals. For example, a silver layer50 nm thick produces an SPR spectrum that is roughly 2 degrees wide.

[0008] The same layer when properly coated produces two different PWRspectra with the two light polarizations, that are more than an order ofmagnitude sharper. The unique characteristics of PWR allow moreinformation about the sample properties to be obtained at much highersensitivities. In particular, probing optically anisotropic samplesrequires the capabilities that PWR offers. Thus, for anisotropicsamples, the refractive index and extinction coefficient have differentvalues for polarizations parallel and perpendicular to the sample plane,yielding information about molecular orientation within the sample.

[0009] See e.g., U.S. Pat. No. 5,521,702—Salamon, et al.—which disclosesthe use and formation of a biocompatible film composed of aself-assembled bilayer membrane deposited on a planar surface inconnection with SPR techniques. See also, U.S. Pat. No.5,991,488—Salamon, et al. which discloses a prism having a metallic filmcoated with a dielectric layer used to provide a surface plasmon wave.

[0010] Most SPR instruments do not record the SPR spectra but reduce theinformation to only the relative angle at which the resonance peak isdetected. This approach eliminates the possibility of determiningoptical properties. Changes in the relative angle are assumed tocorrelate to changes in the refractive index of the sample layer(measured with only one polarization) due to mass moving into and out ofthe layer. For this to be true, it is assumed that the sample isisotropic and that the thickness and absorbance (or scattering) areconstant. Unfortunately these assumptions are not always correct inpractical applications and can result in misleading data and erroneousconclusions. In addition, the molecular interactions resulting inchanges in mass of the sample also usually influence the molecularorganization. As an example, conformation changes occurring without netbinding will result in changes in the relative angle. Further, changesin the bulk solvent will produce changes in the relative angle and canappear as binding effects. One way to avoid such misleading measurementsis to use both polarizations and to analyze the full resonance spectrum.

[0011] There are a number of applications for a PWR spectrometer. Forexample, PWR devices can be used to probe molecular interactions (i.e.binding followed by structural alterations induced by binding) withinanisotropic interfaces and thin films, including: optical coatings,lipid bilayers, proteins and peptides inserted into lipid bilayers, andothers. It can also be used the way as a conventional SPR instrument tofollow changes in the angular resonance peak position.

[0012] The invention is directed to improvements in membrane formationtechniques as well as improvements in automation techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a pictorial view of a PWR device for use in accordancewith the invention.

[0014]FIG. 2 is a pictorial view showing a more detailed view of aturntable, support block and prism in accordance with FIG. 1.

[0015]FIG. 3 is a pictorial view showing a more detailed view of theprism, support block, sample well, ports and passages in accordance withFIG. 2.

[0016]FIG. 4 is a pictorial view showing an exemplary prism structurefor use in accordance with the invention.

[0017]FIG. 5 is a pictorial view showing the prism structure of FIG. 4and a sample layer in accordance with the invention.

[0018]FIG. 6 is a top view pictorial diagram showing the aiming of asyringe used to form a membrane in accordance with the invention.

[0019]FIG. 7 is a side view pictorial diagram showing the aiming of asyringe used to form a membrane in accordance with the invention.

[0020]FIG. 8 is a pictorial view of a support block for supporting aneedle in accordance with the invention.

[0021]FIG. 9 is a pictorial view of a support block for supporting aneedle in accordance with the invention.

[0022]FIG. 10 is an exemplary diagram showing the interconnection ofvarious system components in accordance with the invention.

[0023]FIG. 11 is a graph showing exemplary graph of raw reflectanceplotted against selected angle for two different layers under test inaccordance with the invention.

[0024]FIG. 12 is a graph showing exemplary graph of reflectance plottedagainst selected angle for various layers under test in accordance withthe invention.

DETAILED DESCRIPTION OF THE INVENTION

[0025]FIG. 1 shows a pictorial view of a surface plasmon resonancedevice used in connection with the invention. The device includes anoptical element with an optical surface upon which a membrane willultimately be formed. In this example, the optical element is a prism 10having an optical surface at least partially coated with thin metalliccoating. For PWR applications, the prism also includes a dielectriccoating at least partially covering the metallic coating. It isunderstood that various optical elements including but not limited toprisms, mirrors, lenses and optical fibers are compatible with theinvention.

[0026] A sample well is formed generally adjacent to the optical surface(in this example, the coated surface of the prism). The sample wellgenerally supports a layer under test, for example a sample layer 12(see FIG. 5). A layer under test can include one or more individuallayers (e.g., self-assembled bilayer membrane). The invention isprimarily directed towards systems and methods for forming anappropriate membrane on an optical surface (in this example the rearsurface of a prism in a SPR or PWR system).

[0027] The layer under test generally forms a test plane that isgenerally adjacent and parallel to the coated surface of the prism. Theprism is mounted to a turntable 14 having an axis of rotation. Thedevice also includes a light source that is generally directed towards asurface of the prism and a detector 18 (see FIG. 2) that detectsreflected and/or scattered light. In a basic mode of operation, a samplelayer is formed in the sample well and at least a portion of the samplelayer is illuminated via the light source. The turntable is rotated anda resonance peak is detected by the detector. The selected angle betweenthe rear surface of the prism and the intensity of the reflectedincident light beam is recorded and is generally correlated with changesin the refractive index, thickness and light absorption of the layer orsample under test.

[0028] Referring to FIG. 1, an exemplary light sources includes red andgreen lasers 20, 22, operable to generate red and green laser beams.Chopper 24 is operable to select one or both laser beams from lasers 20,22. Mirrors 26 direct the selected laser beam sources towards beamcombiner 28. The resultant red, green or mixed (e.g., orange) light isthen directed towards mirror 27 and then polarizer 30 such that anappropriate polarizer is optionally selectable (e.g., linearpolarizer—vertical or horizontal). It is understood that a broad rangeof light wavelengths can be used (e.g., ultraviolet, infrared etc. etc).

[0029] The light beam is then directed towards a beam splitter whichdivides the light beam into essentially two portions. The first portionof the light beam is directed towards a reference detector. The secondportion of the light beam (i.e., source beam) is directed towards amirror 40 and finally the source beam is directed towards the prism 10.One or more pumps 42 with associated actuators, syringes and electronicsare provided as discussed in more detail below.

[0030] Preferably, various system components (e.g., turntable, pumps andthe like) are coupled to processor 100, operable to automate varioussystem functions as discussed below. Processor 100 can be implementedusing a typical personal computer and associated operating system suchas a Microsoft Windows product, Linux or the like. The hardware andsoftware configuration of a processor operable to control and automate aPWR device in accordance with the invention based on the disclosureherein is well within the grasp of those skilled in the art.

[0031] The term “light source” as recited herein refers to source oflight in its broadest sense. It is understood that a variety ofdifferent light sources can be used to produce a suitable light beam,including but not limited to, semiconductor lasers, gas lasers, solidstate lasers and the like. It is also understood that a light source caninclude various intermediate devices, including but not limited to,optical fibers, lenses and/or mirrors for focusing, collimating,polarizing, filtering, aiming and/or altering the properties of thelight beam. Accordingly, the term “light source” as recited herein isnot limited to the precise arrangement shown in FIG. 1.

[0032]FIG. 2 shows a more detailed view of the turntable 14 and prism10. As discussed above, turntable 14 has an axis generally located inthe center of the turntable. Preferably, turntable 14 is coupled to arotary drive mechanism (not shown) operable to automate rotary movement.Preferably, the rotary drive mechanism is coupled to a controller and/orprocessor (see e.g., FIG. 1—processor 100) operable to initiate rotarymovement of the turntable and track the angle of the turntable withrespect to the source beam. The tracking of selected angles,interconnection of turntables, rotary drive mechanisms and controllersand/or processors based on the disclosure herein is well within thegrasp of those skilled in the art.

[0033] Referring to FIGS. 2 and 3, a support block 150 is generallypressed against the rear surface of the prism. The support block isgenerally formed with at least one sample well 152 (see FIG. 3). Thesupport block is also preferably formed with one or more ports 154 eachhaving at least one associated passage 156 (see FIG. 3). The ports 154and passages 156 are generally utilized to carry fluids (e.g., liquidsor gasses) to and/or from the sample well 152, as discussed on moredetail below. Support block 150 can be fabricated from a variety ofmaterials. In the current example, the support block is preferablyformed from PTFE (e.g., TEFLON®).

[0034]FIG. 3 shows only a sectional view of the support block 150, asingle sample well 152 and a single port 154 with one associated passage156. It is also understood that a plurality of ports can be provided,each having one or more passages. It is understood that the sample blockcan be formed with a plurality of sample wells, each having one or moreassociated ports and passages. In the current example, the rear surfaceof the prism is disposed generally vertically. Accordingly,gravitational forces must be taken into account with respect to thevarious port locations. For example, ports intended to deliver fluids tothe sample well can have an opening located in the upper portion of thesample well. Ports intended to remove fluids from the sample well canhave an opening located in the lower portion of the sample well.

[0035]FIGS. 4 and 5 show an exemplary prism structure for use inaccordance with the invention. The support block has been omitted forpurposes of clarity. Sample layer 12 is shown generally located on thecenter of the rear surface of the prism. It is understood that samplelayer 12 can be formed on any portion of the rear prism surface. It isalso understood that in the current example, sample layer 12 is at leastpartially surrounded by a sample well formed in an associated supportblock.

[0036] The prism 10 has an entrance surface 60, an exit surface 62 and arear surface 64. In the current example, the rear surface of the prismis at least partially coated with a metallic film 66. In the case of PWRapplications, the prism is optionally coated with a dielectric layer 67(e.g., silica dioxide). As discussed above, the two metals that producethe strongest SPR signals are gold and silver. Since PWR techniquesinclude a dielectric layer, silver is preferable. However, othermetallic layers are compatible with the invention.

[0037] Membrane Formation

[0038]FIGS. 6 and 7 show the relationship between a syringe and thesample well and the rear surface of the prism during membrane formation.The support block is shown in sectional form. The sample well 152 isgenerally circular in cross section, thereby defining an open cell withPTFE walls and a generally vertically disposed circular rear cellsurface (i.e., the rear surface of the prism). The PTFE walls aregenerally sloped as shown in FIGS. 2, 6 and 7. It is understood thatvarious geometric profiles and surface textures can be used withoutdeparting from the invention. The membrane will be formed between thePTFE walls and will rest on the rear cell surface (i.e., on the rearsurface of the prism—over the metal film and/or dielectric if suchlayers are present).

[0039] In this example, the diameter of the circular rear cell surfaceis approximately 5 mm. The syringe 158 volume is 10 microliters and isfitted with a 0.13 mm needle (inside diameter). Syringe 158 is filledwith a suitable membrane solution (e.g., egg phosphatidlcholine insqualene and butanol). Syringe 158 is generally positioned in front ofthe cell and the needle 157 is aimed approximately 1 mm above the centerof the cell. It is understood that the syringe can be supported by avariety of different structures. In this example, the syringe isdisposed at an angle δ with respect to the rear surface of the prism. Inthe current example, δ is in the 20°-40° range.

[0040]FIGS. 6 and 7 show a syringe 158 with a needle coupled directly tothe syringe body. It is understood that intervening structures such asconduits or tubing can be used to couple the needle and syringe body influid communication. To this end, FIGS. 8 and 9 show an exemplarysupport block 150 with a membrane formation port 155 operable toposition and aim a needle appropriately. The port is coupled to thesyringe via a conduit (not shown). It is understood that the termsyringe herein encompasses a variety of structures operable to dispensea metered quantity of fluid without departing from the scope of theinvention.

[0041] The plunger 159 is rapidly depressed such that a steady stream ofmembrane solution is ejected from the needle and strikes the circularrear surface of the cell. A membrane is formed on the rear cell surface.It is understood that syringe 158 can be coupled to a suitable pump(e.g., one of pumps 42 shown in FIG. 1) so that membrane formation canbe initiated under processor control.

[0042] The membrane has a substantially uniform thickness and covers theentire rear cell surface. Coverage of the rear cell surface is difficultto achieve because the membrane solution is hydrophobic and willpreferentially interact with the hydrophobic PTFE walls of the supportblock 150. When the membrane solution is injected on the rear cellsurface (e.g., coated rear surface of the prism) it should spreaduniformly to make contact with the PTFE at the perimeter of theavailable coated surface. When the coated rear surface is in a verticalposition the initial contact point of the membrane solution must beabove the center point of the coated rear surface (as described by thecircular opening in the PTFE support block) to offset the effect ofgravity.

[0043] The invention is advantageous in that a membrane is created on anoptical surface while it is located in the operating position of theinstrument or system. This aids in maintaining the position of thesurface relative to the other instrument components to a tight tolerance(e.g., better than millidegrees of rotation). Prior to the invention,the formation of such a membrane required disassembly of the apparatusfor manual application of the membrane solution. See e.g., U.S. Pat. No.5,521,702—Salamon et al. The invention is also advantageous in that themembrane formation as well as other system functions can be automated.

[0044] To this end, FIG. 10 is an exemplary diagram showing theinterconnection of various system components. Syringe #1, Syringe #2 andSyringe #3 are coupled to pumps 42 (FIG. 1) and are generally operableto dispense a metered quantity of fluid. Pumps 42 generally include asyringe support and a linear slide that engages with the syringeplunger. The linear slide and associated actuator that are preferablycoupled to a processor (e.g., processor 100). The hardware and softwareconfiguration of a processor operable to control and automate a pump inaccordance with the invention based on the disclosure herein is wellwithin the grasp of those skilled in the art.

[0045] Syringe #1, syringe #2 and syringe #3 are coupled to valves 162,164 and 166 via conduits. Valve 162 is an 8 way valve operable to couplesyringe #1 to a plurality of different reservoirs (e.g., 170—Waste,172—N₂, 174H₂O, 176—Buffer and 178 Titrant). Valves 164 and 166 aretypical three way L port valves. Valve 164 is operable to selectivelycouple syringe #2 to either reservoir 180 (H₂O) or port #8 on thesupport block 150. Valve 166 is operable to selectively couple syringe#3 to either reservoir 182 (buffer) or port #2 support block 150.

EXAMPLE Running a Lipid PWR Experiment

[0046] In this example, the support block is formed with three samplewells and the rear surface of the prism is partially coated with ametallic coating, thereby defining a coated portion and an uncoatedportion. A first sample well 190 is located on an uncoated portion ofthe prism (bare reference position). A second sample well 192 is locatedon a coated portion of the prism (sample position). A third sample well194 is also located on a coated portion of the prism (coated referenceposition). The device is generally operable to direct the light source50 towards any of the sample wells under processor control. Thestructures required to shift the source beam to the desired position onthe rear surface of the prism are disclosed in a U.S. patent applicationfiled Jul. 26, 2002, entitled “Beam Shifting Surface Plasmon ResonanceSystem and Method” (Attorney Docket No. 367264-102)—herein incorporatedby reference in its entirety. It is also understood that all of thevarious system components can be coupled to a processor operable toinitiate and coordinate operation of the components (e.g., operation ofvalves, extraction of fluids from reservoirs and delivery of fluids tothe sample well).

[0047] The following is an example of how and experiment can beconducted using the disclosed system.

[0048] 1. Turn on instrument and software to start warming up.

[0049] 2. Clean support block 150 and prism 10 and tubing.

[0050] 3. Prepare lipid and buffer:

[0051] A. 8 mg/ml egg PC dissolved in squalene/butanol (0.007:0.993;v/v)

[0052] i. Open one 50 mg vial after tapping lipid into bottom.

[0053] ii. Add to vial 6.21 ml of butanol and 44 μl of squalene.

[0054] iii. Mix until lipid is dissolved, transfer to screw capped tube.

[0055] B. 10 mM TRIS, 10 mM KCl, 0.5 mM EDTA

[0056] i. 154.6 mg TRIZMA HCl

[0057] ii. 75 mgKCl

[0058] iii. 19 mg Disodium EDTA

[0059] iv. Mix in 100 ml of DI water and adjust to pH 7.3

[0060] 4. Assemble block, prism and tubing.

[0061] 5. Calibrate Baseline with Air as the reference at the BareReference Position.

[0062] 6. Fill Sample and Bare Reference Positions with fresh pureWater:

[0063] A. Using valve 1 to fill Sample, lines 5 and 7, then fill line 1until no bubbles come out line 3.

[0064] B. Use valve 2 to fill Bare Reference until no bubbles come out.

[0065] 7. Calibrate Angle with Water as the reference at the BareReference Position.

[0066] 8. Take Water spectra at the Sample Position over the 60 to 70degree range:

[0067] A. Red P

[0068] B. Red S

[0069] C. Green S

[0070] D. Green P

[0071] 9. Fill Sample lines 5 and 7 with air, drain line 1 to waste.

[0072] 10. Dry lines 5, 7 and 1 with nitrogen, exiting through line 3.

[0073] 11. Remove line 5 and inject 1.5 μl of lipid.

[0074] 12. Fill Sample and Bare Reference Positions with fresh buffer:

[0075] A. Using Syringe and Valve 162 to fill Sample Position, lines 5and 7, then fill line 1 until no bubbles come out line 3.

[0076] B. Use Syringe and Valve 2 to fill Bare Reference Position untilno bubbles come out.

[0077] 13. Take the Red S spectrum at the Sample Position for initialconditions.

[0078] 14. Take Buffer TIR spectra at Bare Reference Position:

[0079] A. Red P

[0080] B. Red S

[0081] C. Green P

[0082] D. Green S

[0083] 15. Repeatedly take Red S spectra at Sample Position at 10 minuteintervals to monitor development of the lipid bilayer. Move to next stepafter spectra are stable. Stability is defined as 0 degree change over{fraction (1/2)} hour.

[0084] 16. Take Lipid spectra at the Sample Position over the 60 to 70degree range:

[0085] A. Red P

[0086] B. Red S

[0087] C. Green P

[0088] D. Green S

[0089] 17. The final Red S spectrum at the Sample Position over therange of 60 to 70 degrees to confirm stability of sample.

[0090] 18. Flush lines with water, then dry with nitrogen.

[0091] 19. Remove and clean Teflon block and prism.

[0092]FIG. 11 is an exemplary graph produced in accordance with theinvention. In this example, raw reflectance of bare reference is plottedagainst the selected angle for two different layers under test (waterand a buffer). FIG. 12 is yet another is an exemplary graph produced inaccordance with the invention. In this example, reflectance of thesample position is plotted against the selected angle for various layersunder test. The left most curve (water) shows a null at approximately64.131° The remaining curves (various lipid bilayers) have nulls thatfalling approximately in the 64.26° to 64.42° range. The results shownin FIGS. 11 and 12 generally illustrate the level of precision that isrealized utilizing the structures and methods disclosed herein. A moredetailed analysis of these graphs is beyond the scope of thisdisclosure.

[0093] While this invention has been described with an emphasis uponpreferred embodiments, it will be obvious to those of ordinary skill inthe art that variations in the preferred devices and methods may be usedand that it is intended that the invention may be practiced otherwisethan as specifically described herein.

What is claimed is:
 1. A spectroscopy system for characterizing surfacephenomenon comprising: at least one light source operable to generate asource beam, an optical element having an optical surface, a supportblock formed with at least one sample well having a center, the sourcebeam being aimed at the sample well, the support block being disposed onthe optical surface thereby defining a substantially vertical rear cellsurface having a center, a syringe filled with a membrane solution influid communication with a needle having a distal end disposed in frontof the sample well, the distal end being aimed at a point above thecenter of the rear cell surface, the syringe be operable to eject asteady stream of membrane solution from the needle onto the circularrear cell surface thereby forming a membrane defining at least a portionof a layer under test, the membrane having a substantially uniformthickness that covers substantially the entire rear cell surface, and adetector operable to detect light that is at least one of reflected andscattered by the layer under test.
 2. The system of claim 1 comprising:at least one actuator coupled to the syringe and a processor coupled tothe actuator wherein the processor is operable to initiate the formationof the membrane.
 3. The system of claim 1 wherein the optical element isat least one of a prism, mirror, lens and optical fiber.
 4. The systemof claim 3 wherein optical surface is at least partially coated with ametallic coating.
 5. The system of claim 4 wherein the metallic coatingis at least partially coated a dielectric layer.
 6. The system of claim1 comprising: a plurality of syringes each having at least oneassociated actuator and a processor coupled to the actuators wherein theprocessor is operable to initiate the delivery of fluids to the samplewell.
 7. A method of forming a membrane in a spectroscopy systemcomprising: providing an optical element having an optical surface,providing a support block formed with at least one sample well having acenter, the support block being disposed on the optical surface therebydefining a substantially vertical rear cell surface having a center,providing a syringe filled with a membrane solution in fluidcommunication with a needle having a distal end disposed in front of thesample well, aiming the distal end being at a point above the center ofthe rear cell surface, ejecting a steady stream of membrane solutionfrom the needle onto the circular rear cell surface thereby forming amembrane defining at least a portion of a layer under test, the membranehaving a substantially uniform thickness that covers substantially theentire rear cell surface.
 8. The method of claim 7 comprising: providingat least one actuator coupled to the syringe and a processor coupled tothe actuator wherein the processor is operable to initiate the formationof the membrane.
 9. The method of claim 7 wherein the optical element isat least one of a prism, mirror, lens and optical fiber.
 10. The methodof claim 9 wherein optical surface is at least partially coated with ametallic coating.
 11. The method of claim 9 wherein the metallic coatingis at least partially coated a dielectric layer.
 12. The method of claim7 comprising: providing a plurality of syringes each having at least oneassociated actuator and a processor coupled to the actuators wherein theprocessor is operable to initiate the delivery of fluids to the samplewell.